The synthesis of polynucleotides is rapidly assuming commercial and industrial importance. Polynucleotides, comprising long polymeric chains consisting of deoxyribose or ribose sugar units linked to purine or pyrimidine base units, and joined into polymeric chains through phosphate linkages, are the chemical basis of nucleic acids. Segments of deoxyribonucleic acid (DNA), i.e. polynucleotide sequences, constitute genes, having encoded therein genetic information related to the sequence of the base units disposed along the polynucleotide chain. Recently, techniques have been developed for linking synthetic polynucleotide sequences into natural DNA, in bacterial cells and the like, so as to modify the genetic information of the cell, and hence to vary the chemical operation of the cell and the products which it is capable of producing. For example, the insulin producing gene has been totally synthesized, and this can now be spliced into bacterial cells to enable the cells to produce greater quantities of insulin by means of their ordinary biological function.
The structural units of DNA are deoxyribose sugar rings to which are linked one of the four purine or pyrimidine bases thymine, guanine, cytosine and adenine. Synthetic processes for making synthetic genes for splicing to DNA must involve the step by step coupling together of each of these four different units, in the desired, predetermined sequence. It is extremely important that the base sequences on all of the molecular chains be exactly correct, or genetic misinformation will be encoded into the synthetic gene, with potentially disastrous results when the gene is spliced into DNA in a cell.
Chemical synthesis of polynucleotides is a multistep, lengthy chemical process. In addition to individual chain extension steps, in which the next nucleotide unit of the sequence is coupled to the polynucleotide chain, the nucleotide reagents and the growing chain must be appropriately chemically protected to ensure that chemical reaction takes place at the correct location on the molecule. Additional process steps of protection and deprotection are therefore necessary.
There has recently been proposed (see U.S. patent application Ser. No. 06/149,685 Kelvin K. Ogilvie and Robert Bender, filed May 14, 1980) a solid state polynucleotide synthesis process, based upon solid polymer support for the growing polynucleotide chains. In this process, a first nucleotide unit is initially condensed with a modified, derivatized solid polymer such as silica gel, to form a coupled initial unit. Then the polymer-nucleotide intermediate is terminally deprotected, and the next nucleotide unit for the predetermined sequence is added to the polymer. These steps are repeated, to build up a polynucleotide chain of desired sequence and length. The process shows significant advantages for large scale operation, in that it can be conducted in a semi-automatic, semi-continuous fashion, with the polymer-polynucleotide growing chains as a solid column, and addition of reagents, solvents and the like sequentially to the column under predetermined conditions and for predetermined lengths of time, in sequence, to build the desired polynucleotide chain.
With the aforementioned polymer support-solid phase process, it is still very necessary to ensure that there is no cross contamination of reagents during the individual process steps. For example, if residues of the thymine-containing nucleotide are present in the reactor column during the stage of reaction when a cytosine nucleotide is supposed to be coupled to the growing polynucleotide chain, then some small proportion of the final polynucleotide chains will have incorrect sequences, and hence will encode incorrect genetic information. It is therefore necessary to provide a synthesis apparatus which substantially eliminates the possibility of any such cross contamination, even when emergency conditions arise, for use in preparation of synthetic genes.